JP2010510986A - TSLP vaccine for treatment of TH2-mediated inflammatory symptoms - Google Patents

Abstract

The present invention relates to therapeutic compositions for TH2-mediated inflammatory conditions. This composition is a vaccine consisting of non-primate TSLP or a fragment thereof and is used as a vaccine for the treatment of humans who have developed inflammatory symptoms mediated by excess TH2. For the treatment of TH2-mediated inflammatory conditions in mammals other than humans, TSLP of the animal species to be vaccinated is used as an antigen.

Description

  The present invention relates to a method for reducing TH2-mediated inflammatory symptoms or a method for suppressing the induction of inflammation by targeting the cytokine TSLP (thymic stromal lymphopoietin). While the present invention relates generally to vaccines for use in mammals, preferred embodiments of the invention relate to vaccines for use in humans, dogs, cats, or horses. In the following, the invention will be described generally with reference to vaccines for humans, cats, horses or dogs.

  In the past decades, several diseases caused by immune system dysfunction have become a major challenge in modern medicine. One such disease is allergic disease. Allergies in humans are almost epidemic in the last 20-30 years, with the most notable diseases being atopy or immunoglobulin (IgE) mediated allergies. Allergies are equally a serious disease for many domestic animals such as dogs, cats and horses. However, few reports have been made on the involvement of IgE in relation to these domestic animals. Common atopic allergies in the case of humans include fur allergy, hay fever, dust mite allergy, insect venom allergy, exogenous asthma, and a wide variety of food allergies. In addition, many domestic animals are allergic to the same type of allergen. It is estimated that nearly 30% of the total human population suffers from allergies.

  Allergic diseases are caused by immune system dysfunction. Several cytokines that regulate normal immune responses against various pathogens are thought to be directly involved in the process of allergic diseases. Cytokines or growth and differentiation factors that play an important role in the regulation of the human immune system are potential targets for disease intervention.

  One particularly difficult problem in the animal medicine field is severe atopic dermatitis in dogs. Almost 50% of the owners who visit veterinarians are those who have skin diseases mainly due to such atopic dermatitis. On the other hand, in humans, the serious medical condition is severe asthma. In extremely severe cases, any existing treatment does not provide a sufficient clinical effect. In the case of dogs, many dogs must be killed in a situation with severe atopic dermatitis. Here is one major medical need that has not yet been addressed.

  For several years, the inventors have studied possible treatment methods for IgE-mediated allergy in humans and domestic animals, and vaccination (injection) against IgE (see references 1-5). However, when IgE levels are exceptionally high, such as in dogs, this treatment method has obvious limitations (see reference 6). Such limitations are mainly due to the strong chemical resistance caused by high concentrations of circulating IgE. Furthermore, it is difficult to obtain a good clinical effect when a large amount of target molecules must be removed. Therefore, in the case of dogs, it has been found that it is almost impossible to achieve our goal by vaccination against IgE (ref. 6). Therefore, the development of new innovative methods is required. The present invention provides one possible solution to the above problem, namely vaccination (injection) against thymic stromal lymphopoietin (TSLP), one of the key cytokines that regulate human immunity.

  TSLP (thymic stromal lymphopoietin) has been described as a master switch for allergic inflammation (Reference 7). TSLP is an IL-7-like cytokine produced mainly by epithelial cells and mast cells in humans. This cytokine stimulates mDC and promotes the differentiation of CD4 + cells that have not undergone drug treatment into TH2-type effector cells [see Reference 7]. These TH2 cells produce allergy promoting cytokines IL-4, IL-5, IL-13 and TNF-α, but not IL-10 and IFN-γ (Reference 8). Recently, DCs activated by TSLP have been found to play an important role not only in TH2 sensitization but also in the maintenance and polarization of TH2 central memory cells in allergic diseases (Reference 9). It has been shown that high levels of TSLP are expressed by keratinocytes in the lesioned part of atopic dermatitis, and that these keratinocytes are the main cytokine during the progression of atopic dermatitis (Reference 8). . Mice genetically engineered to express excess TSLP in the skin also develop atopic dermatitis, which causes a dramatic increase in circulating TH2 cells and an increase in serum IgE (Reference 10). However, TSLP is expressed not only by peripheral tissues but also by Hassall bodies in the human thymus. In this case, TSLP is involved in thymic DC commands and transforms high affinity autoreactive T cells into CD4 + CD25 + FoxP3 + regulatory T cells. With respect to that receptor, the heterodimeric TSLP receptor appears to be expressed exclusively by bone marrow dendritic cells (mDC).

TSLP appears to be one of the key regulators of TH2-mediated inflammatory symptoms and may therefore be a very important target for therapeutic intervention (Ref. 11). The following describes a vaccination method (injection) aimed at alleviating excessive TH2-mediated inflammatory symptoms by targeting TSLP. This method of vaccination can be a new and important step in the treatment of severe asthma in humans and in the treatment of atopic dermatitis in dogs.
To date, patent applications have been filed describing the use of monoclonal antibodies and water soluble receptors that block the activity of human TSLP. These methods of targeting TSLP rely on injecting highly purified recombinant proteins every 2-4 weeks over the life of the patient. In the case of the vaccine described here, the dependence on recombinant protein injection is considerably less than in the prior art, perhaps 10,000 compared to the amount required for treatment with monoclonal or water-soluble receptors. It is greatly improved in that it is reduced to about twice. In most cases, this vaccine only needs to be administered 1 to 4 times a year, whereas in the case of the monoclonal or water-soluble receptors mentioned above, it needs to be administered more frequently. The use of non-human primate sequences may increase the vaccine immunity that occurs in humans. If a therapeutic method other than a vaccine is used, an unaltered TSLP (human protein) should be used so that an immune response against the protein that significantly reduces the effectiveness of the injected recombinant protein is not elicited. is there.

Hellman L. “Reduced allergen sensitivity after treatment with a new allergic vaccine”, Eur J Immunol 1994; 24 (2): 415-20. Hellman L. “Is a vaccine against IgE possible?”, Adv Exp Med Biol 1996; 409: 337-42. Hellman L, Carlsson M. "Allergy Vaccines, Review of Development", Clin Immunotherapeutics 1996; 6 (2): 130-42. Hellman L. “Vaccines against allergies”, In: Perlmann P, edited by Wigzell H, Handbook of Experimental Pharmacology, Vol 133, Vaccines. Berlin, Springer-Verlag, 1999: 499-526. Vernersson M, Ledin A, Johansson J, Hellman L. "Induction of therapeutic antibody response to IgE", Faseb J 2002; 16 (8): 875-7. Ledin A, Bergvall K, Salmon-Hillbertz N, Hansson H, Andersson G, Hedhammar A, et al, "Induction of therapeutic antibody response to IgE in dogs as an animal species with exceptionally high plasma IgE levels", Vaccine 2006 ; 24, 66-74. Liu YJ. “Thymic stromal lymphopoietin: the master switch of allergic inflammation”, J Exp Med 2006; 203 (2): 269-73. Soumelis V, Reche PA, Kanzler H, Yuan W, Edward G, Homey B, et al. "Human epithelial cell-triggered dendritic cell-mediated allergic inflammation by TSLP generation", Nat Immunol 2002; 3 (7): 673-80. Wang YH, Ito T, Wang YH, Homey B, Watanabe N, Martin R, et al. "Maintenance and polarization of human TH2 central memory T cells by thymic stromal lymphopoietin-mediated dendritic cells", Immunity 2006; 24 (6): 827-38. Yoo J, Omori M, Gyarmati D, Zhou B, Aye T, Brewer A, et al. "Especially mouse spontaneous atopic dermatitis expressing thymic stromal lymphopoietin conversion gene inducible in skin", J Exp Med 2005; 202 (4): 541-9. Huston DP, Liu YJ. “Thymic stromal kenfopoietin: a potential therapeutic target for allergies and asthma”, Curr Allergy Asth ma Rep 2006; 6 (5): 372-6.

  The present invention aims to provide a simple and cost-effective method for treating various inflammatory conditions caused by excessive TH2-type immunity. Treatment with a vaccine comprising a fusion protein consisting of TSLP (or a portion of TSLP) and a foreign carrier protein reduces free TSLP levels, thereby alleviating symptoms caused by excessive release of cytokines.

  The above object is based on the entire TSLP amino acid sequence obtained from a non-human primate (when used in humans) provided by the present invention, or a segment consisting of more than 5 amino acids of said amino acid sequence. Or a vaccine that includes proteins in multiple conjugated forms. When processing a domestic animal, a TSLP sequence obtained from the animal species to be vaccinated is used instead of the vaccine. The protein can optionally be bound to one or more heterologous carrier proteins by including an adjuvant. TSLP molecules used in vaccine antigens can be altered by mutagenesis in one or several places to block their binding to the receptor. This maintains the protein's ability to induce antibodies that react with native TSLP, although the protein has no TSLP activity when injected into the host.

It is the figure which showed the nucleotide and corresponding amino acid sequence of chimpanzee TSLP.

Means for carrying out the invention

Active immunization, ie vaccination (injection), generates anti-TSLP antibodies in the host. By inoculating (injecting) the modified TSLP molecule into the host, the host immune system causes a polyclonal antibody response against its own TSLP, thereby down-regulating the effects of potentially excessive TSLP production. It is most important to alter the antibody so that the host immune system recognizes the modified self protein as a non-self protein. To achieve this, it is necessary to covalently link the non-self amino acid moiety to TSLP or to a specific part of TSLP obtained from the animal species to be treated. This attracts or activates non-tolerant T cells that help the function of potentially self-reactive B cells by the peptides in the non-self part.
There are at least four ways to modify self-proteins. One is to generate a fusion protein between a non-self protein and a whole fragment of self-TSLP or a fragment of more than 5 amino acids in a prokaryotic or eukaryotic expression system. Then, an open reading frame of TSLP, exemplified by the dog and human TSLP of FIG. 1, is first cloned into a bacterial, mold or eukaryotic fusion protein vector. This fusion protein construct is then transfected into a mammalian or prokaryotic host to produce the desired fusion protein. In this case, the fusion object can be any non-self protein consisting of 10 amino acids or several hundred kD in size. However, it is generally preferred to use a fusion object having approximately the same size as the self protein.

  Alternatively, as a second alternative, the immunodominant peptide can be inserted into the TSLP structure to produce a fusion protein with a self TSLP sequence on both sides of the foreign peptide.

  As a third method, unmodified TSLp can be produced in a mammalian host, or prokaryotic host or host cell line, and then covalently bound to a carrier protein by chemical linkage.

  The fourth method, which is slightly less preferred, is a method in which specific portions of the TSLP sequence are generated as synthetic peptides, and these peptides are then bound to foreign carrier molecules by chemical bonds. This fourth method usually results in an antibody response that, after injection into a patient, exhibits a low binding activity to the native, properly folded protein and thus has a low clinical effect.

  After manufacture, the vaccine antigen is then purified and examined for pyrogen content and possible other contaminant content. Optionally, to obtain a sufficiently strong immune response against the self epitope, the vaccine antigen is then mixed with an adjuvant before being injected into the patient. After administration to a patient, the vaccine induces an immune response against the vaccine antigen. Due to the presence of self epitopes in the vaccine antigen, this protein also induces an antibody response to the target molecule, in this case TSLP, thereby reducing the level of this protein present in the patient.

Claims (6)

  A vaccine comprising TSLP or at least one fragment thereof obtained from a non-human primate or other non-human mammal and a pharmaceutically acceptable adjuvant.   2. A vaccine according to claim 1, characterized in that at least one fragment of TSLP is bound to a carrier molecule in its original form or in multiple conjugated form.   The vaccine according to claim 1 or 2, wherein the TSLP is a TSLP obtained from a non-human primate, that is, a dog, a cat, or a horse.   Use of TSLP vaccine as a medicine.   Generating a cDNA or TSLP genomic sequence, or a portion of TSLP encoding more than 5 amino acids as a clone, ligating the generated clone into a suitable vector, eukaryotic or prokaryotic host cell To produce a fusion protein, to include the entire TSLP sequence or part thereof into the original form of TSLP, mutated TSLP, or multiple conjugated TSLP, and the resulting fusion protein The method for preparing a vaccine according to claim 1, characterized in that it comprises the steps of purification and optionally mixing with a suitable adjuvant.   Use of a fusion protein consisting of all or part of TSLP obtained from an animal species to be treated (or a closely related animal species) and a foreign carrier protein for the production of a vaccine used as a medicine.

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